Non-invasive molecular imaging plays a key role in detection of disease by characterizing and measuring biological processes at the molecular level. A number of medical diagnostic procedures, including PET and SPECT utilize radiolabeled compounds. PET and SPECT are very sensitive techniques and require small quantities of radiolabeled compounds, called tracers. The labeled compounds are transported, accumulated and converted in vivo in exactly the same way as the corresponding non-radioactively compound. Tracers or probes, can be radiolabeled with a radionuclide useful for PET imaging. Such radionuclide may include 11C, 13N, 15O, 18F, 61Cu, 62Cu, 64Cu, 67Cu, 68Ga, 124I, 125I and 131I, or with a radionuclide useful for SPECT imaging, such as 99Tc, 75Br, 61Cu, 153Gd, 125I, 131I and 32P.
PET creates images based on the distribution of molecular imaging tracers carrying positron-emitting isotopes in the tissue of the patient. The PET method has the potential to detect malfunctions on a cellular level in the investigated tissues or organs. PET has been used in clinical oncology, for the imaging of tumors and metastases, and has been used for diagnosis of certain brain diseases, as well as for mapping brain and heart function. Similarly, SPECT can be used to complement any gamma imaging study, where a true 3D representation can be helpful, for example, imaging tumor, infection (leukocyte), thyroid or bones.
The formation of new blood vessels sprouting from existing blood vessels, is a fundamental process, known as angiogenesis, associated with tumor progression. Angiogenesis is regulated by a balance between pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and anti-angiogenic molecules, such as angiostatin and endostatin. Most tumors begin growing as avascular dormant nodules until they reach steady-state populations of proliferating and apoptosing cells. Angiogenesis starts with perivascular detachment and vessel dilation, followed by angiogenic sprouting, new vessel formation, maturation, and the recruitment of perivascular cells. Blood vessel formation continues as the tumor grows, feeding on hypoxic and necrotic areas of the tumor for essential nutrients and oxygen. This multi-step process offers several targets for the development of tumor angiogenic and metastatic diagnostics.
Integrins, are largely responsible for cell-cell and cell-matrix interactions, and are one of the main classes of receptors regulating tumor metastasis and angiogenesis. In addition to having adhesive functions, integrins transduce messages via various signaling pathways influencing proliferation and apoptosis of both tumor cells, and activated endothelial cells. Research has shown that integrins are a family of adhesion molecules consisting of two noncovalently bound transmembrane subunits (α and β). Both subunits are type I membrane proteins with large extracellular segments that pair to create heterodimers with distinct adhesive capabilities. In mammals, 18α and 8β subunits assemble into 24 different receptors. One prominent member of this receptor class is the integrin αvβ3 receptor. The special role of integrin αvβ3 in tumor invasion and metastasis arises from its ability to recruit and activate matrix metalloproteinases 2 (MMP-2) and plasmin, which degrade components of the basement membrane and interstitial matrix. It has been demonstrated that tumor expression of integrin αvβ3 correlates well with tumor progression in several malignancies such as melanoma, glioma, breast cancer, and ovarian cancer. The receptor αvβ3 is not readily detectable in quiescent vessels but becomes highly expressed in angiogenic vessels, serving as an excellent molecular marker for tumor metastasis and angiogenesis imaging. Thus, the ability to noninvasively visualize and quantify integrin αvβ3 expression level will provide new opportunities to document tumor integrin expression, to properly select patients for anti-integrin treatment, and to monitor treatment efficacy in integrin-positive patients.
Based on the findings that several extracellular matrix proteins, such as vitronectin, fibrinogen, and thrombospondin interact with integrins via the amino acid sequence arginine-glycine-aspartic acid (RGD). Linear and cyclic peptides containing the RGD sequence have been extensively explored and tested. Kessler and co-workers [1] developed the pentapeptide cyclo(-Arg-Gly-Asp-D-Phe-Val-) (“c(RGDfV)”) which showed both high affinity and selectivity for integrin αvβ3. To date, most integrin αvβ3 targeted PET studies have utilized the radiolabeling of c(RGDfV)-based antagonists due to their high binding affinities which range from nanomolar to subnanomolar range for monomeric and multimeric c(RGDfV) respectively. In particular, most efforts [2-4] are focused on the modification of the linkage connecting cyclic RGD peptide to the radionuclide. Currently, [18F]Galacto-RGD [5-7] represents the most promising integrin marker in the clinical trial arena. Despite its successful translation into clinical trials, several key issues remain to be resolved. As a monomeric RGD peptide tracer, it has a relatively low tumor targeting efficacy. In addition, its clinical utility is severely limited because of its relatively low integrin binding affinity, modest tumor standard uptake values, and unfavorable pharmacokinetic behavior. Therefore, tumors with low integrin expression levels may not be detectable. In addition, prominent tracer accumulation in the liver, kidneys, spleen, and intestines was observed in both preclinical models and human studies resulting in difficult visualization of abdomen lesions. To add to its imaging drawbacks, the synthetic preparation of the tracer is labor intensive, time consuming and inefficient, thereby limiting its widespread availability to clinicians.
Recently, a library of RGD-containing pseudopeptides has been synthesized [8]. These compounds are characterized by the replacement of the D-Phe-Val or the D-Phe-[NMe]Val dipeptide with a 6,5- and 7,5-fused bicyclic lactam. In comparison with D-Phe-Val or D-Phe-[NMe]Val dipeptide, bicyclic lactams show different reverse-turn mimetic properties that constrain the RGD sequence into different conformations and provide the required integrin activity and selectivity. While these cyclic peptides validate the use of conformationally constrained RGD peptides as integrin ligands, they cannot be used directly for PET imaging due to their difficult synthesis.